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Original Studies |
Fetal and Neonatal Stress Research Centre (R.G., N.M.F., J.M.A.T., V.G.), Department of Maternal and Fetal Medicine, Division of Paediatrics, Obstetrics and Gynaecology, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, United Kingdom; and The Queen Mother’s Hospital (A.C.), Yorkhill, Glasgow G3 8SJ, United Kingdom
Address all correspondence and requests for reprints to: Rachel Gitau, Fetal and Neonatal Stress Research Centre, Department of Maternal and Fetal Medicine, Division of Paediatrics, Obstetrics and Gynaecology, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, United Kingdom.
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Abstract |
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=
52.6 nmol/L, 95% CI (25.3–79.9), P = 0.001; mean
ß-endorphin response =106 pg/mL, 95% CI (45.3–167),
P = 0.002]. Baseline maternal fetal ratios were 13
[95% CI (10.7–15.2)] for cortisol and 0.8 [95% CI (0.5–1.0)]
for ß-endorphin. The novel findings were: 1) that the fetal responses
were independent of those of the mother, which did not change during
transfusion at either site; 2) that there was a correlation between
baseline fetal and maternal cortisol levels (r = 0.58, n =
51, P < 0.0001) but not between baseline fetal and
maternal ß-endorphin levels, suggesting cortisol transfer across the
placenta, rather than joint control by placental CRH; and 3) that fetal
ß-endorphin responses were apparent from 18 weeks gestation and
independent of gestational age, whereas fetal cortisol responses were
apparent from 20 weeks gestation and were dependent on gestational age
(y = -91.4 + 5.08x, r = 0.51; n = 16;
P = 0.04; CI for slope, 0.16–10.0), consistent
with the maturation of the fetal pituitary before the fetal adrenal. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Women with singleton pregnancies, undergoing clinically indicated fetal blood sampling or intrauterine blood/platelet transfusion at the Center for Fetal Care, Queen Charlotte’s and Chelsea Hospital or at The Queen Mother’s Hospital, were recruited for the study. The indications for fetal blood sampling were rapid karyotyping or suspected anemia and for intrauterine transfusion, were fetal anemia or thrombocytopenia in alloimmunized pregnancies.
Fetuses were considered suitable for inclusion when the fetus was appropriately grown for gestational age and structurally normal on preprocedural ultrasound, with no evidence of hydrops, absent end-diastolic frequencies in the umbilical artery Doppler waveform, severe anemia (hemoglobin, <5 g/dL), or hypoxia [fetal pO2 below the reference range (5)], and had a normal karyotype, to exclude conditions that can be associated with basal elevations in the study endpoints (6, 7). Fetal sex was also determined during preprocedural ultrasound and confirmed by karyotyping. Complicated procedures, involving multiple fetal vessel punctures, or when time to access the fetal circulation exceeded 10 min [shown to cause elevations in cortisol and ß-endorphin (1)], were excluded. Fifty-one fetuses satisfied the criteria for inclusion. Some of the results with cortisol have been reported previously in correspondence form (3). Twenty-three fetuses satisfied the criteria for the transfusion group.
The site of ultrasound guided fetal blood sampling or transfusion was chosen by the operator on the basis of technical factors and ease of approach, as per usual practice. Randomization of site sampling was not considered appropriate, because anatomical factors often dictate one approach in favor of the other. Neither fetal neuromuscular blockade nor analgesia was used. The mothers did not receive sedation. Intravascular transfusion was performed using packed red blood cells or platelets, warmed to body temperature before transfusion. The purity of fetal samples was confirmed by comparison of fetal and maternal mean corpuscular volumes and subsequent Kleihauer-Betke testing. Full blood count (Coulter Counter, Coulter Electronics, Luton, UK) and blood gases (Radiometer ABL 330 blood gas analyzer, Copenhagen, Denmark) were also analyzed.
Blood samples
Two milliliters of additional venous fetal blood were drawn into a heparinized syringe, after collection of clinical samples, and placed in a chilled heparinized tube. Seven milliliters of maternal blood samples were collected by venepuncture into a heparinized Vacutainer (Becton Dickinson and Co., Meylan Cedex, France) immediately before needling or transfusion, and within 10–20 min after the procedure.
Fifty microliters (500 kIU) of Trasylol/mL blood was added, immediately after collection, to all tubes. Bloods were spun in a refrigerated centrifuge at 3,000 x g to separate plasma, which was stored at -80 C until assay.
Timings
Samples were collected between 1000–1800 h, the majority (90%) between 1200–1700 h. The time from first puncture of the fetal trunk or cord until accessing the fetal circulation and collection of the first blood sample was recorded as: time to access. The duration of transfusion was recorded from the time of access until the taking of the second blood sample at the end of the transfusion. Procedures were timed using a digital stopwatch and were recorded in minutes and seconds. For analysis, times were rounded to the nearest 0.1 min. Procedures with time to access that was greater than 10 min were excluded.
Written informed consent was obtained from all the mothers, for the collection of additional blood samples for research purposes, in accordance with the Institutional Ethics Committee requirements.
Assays
Cortisol levels were assayed using a standard solid-phase RIA (by DPC, Los Angeles, CA), and maternal and fetal plasma sample pairs were analyzed in the same assay run. The lower limit of sensitivity was 10 nmol/L. The intra- and interassay coefficients of variation were 5.3% and 4.3%, respectively. Plasma levels of ß-endorphin were determined using a solid-phase two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The lower limit of sensitivity was 14 pg/mL, and cross-reactivity with lipotropin was 14%. The intra- and interassay coefficients of variation were 13.2% and 9.5%, respectively. In a few cases, there was insufficient sample volume to measure ß-endorphin levels as well as cortisol.
Statistics
All ranges studied were normally distributed, except for fetal ß- endorphins, time to access, and time of transfusion, which were normalized by log transformation. Data were analyzed by standard parametric statistics using SPSS, Inc. 9.0 for Windows (Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There was no significant correlation between basal concentrations
of either fetal or maternal cortisol and gestational age, over the
range studied (17–35 weeks), nor was there with fetal ß-endorphin.
However, maternal basal ß-endorphin rose significantly with advancing
gestational age [y = -17.7 + 2.20x; r = 0.36; n = 37;
P = 0.03; 95% confidence interval (CI) for slope,
0.27–4.13] (Table 2). There was no
correlation between maternal or fetal basal values and time of day.
There was no difference in basal fetal values by sex of the fetus for
either cortisol [mean (95% CI): males = 48.3 (42.2–54.4),
n = 24; females = 52.3 (40.5–64.1), n = 15] or
ß-endorphin [geometric mean (95% CI): males = 72.9
(53.2–99.5), n = 22; females = 74.6 (51.9–104), n =
15].
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). However, fetal
ß-endorphin was unrelated to maternal levels [r = -0.2, n
= 36, not significant (ns) (Fig. 1b)].
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) but not cortisol
(r = 0.2, n = 31, ns) (Fig. 2a). If only procedures
lasting 
6 min were considered, n = 21, the correlation
with time and ß-endorphin disappeared. There was no significant
correlation between time to access the PCI and fetal cortisol or
ß-endorphin levels.
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There was a significant correlation between maternal baseline cortisol and ß-endorphin levels (r = 0.52, n = 35, P = 0.002). There was no such correlation between fetal baseline levels (r = 0.048, n = 46, ns).
Fetal responses
Several fetuses required multiple transfusions at the same site; in these cases, the first transfusion at each site was used.
Pretransfusion cortisol and ß-endorphin levels were similar in the
IHV and PCI groups (Table 1
). Figure 3
shows that transfusion resulted in a significant rise in fetal plasma
cortisol levels when carried out at the IHV [mean , 52.6 nmol/L;
95% CI (25.3–79.9); P = 0.001] but not when the same
procedure was done via the PCI [mean , 3.27 nmol/L; 95% CI (-13.5
to 20.1); P = 0.7]. Similarly, there was a fetal ß-
endorphin response to IHV transfusion [mean , 106 pg/mL; 95%
CI (45.3–167); P = 0.002] but not to PCI transfusion
[mean , 12.2 pg/mL; 95% CI (-11.9 to 36.3); P =
0.3].
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The magnitude of the cortisol response at the IHV increased with
gestational age (y = -91.4 + 5.08x; r = 0.51; n = 16;
P = 0.04; 95% CI for slope, 0.16–9.99); there was no
such correlation between the ß-endorphin response and gestational age
(r = 0.03, n = 14, ns) (Fig. 4
and Table 2).
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Paired pre- and posttransfusion maternal samples were available
for 7 IHV and 5 PCI procedures. Maternal cortisol and ß-endorphin
levels did not change with transfusion [mean s, 28.9 nmol/L (95%
CI, -60.7 to 119) and 5.82 pg/mL (95% CI, -6.08 to 17.7),
respectively (Fig 5)]. values were
similar in IHV and PCI transfusions. This lack of response was equally
apparent at all gestational ages studied (range, 22–35 weeks). There
was no relationship between paired maternal and fetal cortisol or
ß-endorphin.
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Discussion |
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The finding of a positive correlation between basal maternal and fetal cortisol confirms and extends our pilot study (3). There was no similar correlation in ß-endorphin levels. This suggests that the cortisol correlation is unlikely to be caused by joint control via placental CRH release into both maternal and fetal compartments, because then, one would expect other POMC derivatives (such as ß -endorphin) also to be correlated. Indeed, Schulte et al. (8) have shown that CRH that is administered to the mother in the third trimester of pregnancy does not increase maternal cortisol.
It has been suggested that high levels of placental 11ß-hydroxysteroid-dehydrogenase type 2 activity excludes maternal cortisol from the fetus (9, 10). A direct study of maternal-fetal cortisol transfer in the fetal-placental unit before abortion showed that 15% of 3H-cortisol crossed the placenta unmetabolized (11). Our finding is compatible with substantial (80–90%) metabolism of maternal cortisol during passage through the placenta. Because fetal levels are about 13-fold lower than maternal, a rise of 10–20% in maternal levels could still double fetal concentrations. Our results indicate that 33.5% of the variance in fetal cortisol is attributable to maternal levels.
If there is direct transfer of maternal cortisol across the placenta in sufficient concentration to have a functional effect on the fetus, this provides a mechanism by which antenatal maternal stress may affect the fetus. Maternal stress in pregnancy has been shown to be associated with babies with lower birth weight and impaired brain development (12). Numerous animal studies have linked antenatal maternal stress with altered long-term hyperreactive HPA responses in the resultant offspring (13). The effects of maternal stress on the fetus can thus have long-term implications (14). The results presented here suggest that a mechanism of direct transfer occurs in humans, so that maternal stress, which results in elevated cortisol levels, may have a direct effect on development.
The correlation between maternal and fetal cortisol levels, suggesting
that maternal cortisol may be transported to the fetus, raises a
question about the independence of the fetal stress response to IHV
transfusions. However, the fact that maternal levels of cortisol and
ß-endorphin did not change with transfusion to the fetus at either
site (Fig. 5) suggests that maternal responses did not influence the
fetal responses described here. It may seem surprising that mothers did
not show any activation of the HPA axis after what one would expect to
be a stressful procedure for her also (i.e. having a
20-gauge needle inserted under local anesthetic through her abdomen
into the fetal circulation, along with a small procedure-related risk
of miscarriage and fetal death). Indeed these procedures are known to
elicit a noradrenaline response in the mother (2).
However, others have observed a desensitization of the maternal HPA
axis response during pregnancy (8), which may, in part, be
caused by release of large amounts of CRH from the placenta
(15). Even though maternal cortisol levels have been shown
to be increased during pregnancy by stressful experiences (8, 16), it may be that a greater insult is needed to achieve the
same effect as in the nonpregnant state. Alternatively, it is also
possible that the mother was stressed in anticipation of the procedure
and, so, was resistant to further HPA activation by the procedure
itself.
The youngest fetus in the previous study was 23 weeks old
(1), whereas the present study included fetuses at earlier
gestational ages. A fetus at 20 weeks showed a typically large
response. This is of interest because it has been suggested that the
human fetal adrenal cortex cannot synthesize cortisol de
novo before 24 weeks (17). However, the fetus at 18
weeks did not show a cortisol response, but he did show a rise in
ß-endorphin. The finding that the cortisol response was significantly
related to gestational age in the range from 18–35 weeks but that the
ß-endorphin response was not, and was actually negative (Table 2,
Fig. 4), suggests that the ß- endorphin response from the fetal
pituitary may mature before the cortisol response from the fetal
adrenal. However, it must be noted that the significant cortisol
correlation (Fig. 4a) depends on a relatively small number of samples
and should thus be interpreted with caution.
Earlier maturation of the pituitary could explain why basal fetal ß-endorphin levels are higher than those of the mother (maternal/fetal ratio, 0.8), whereas cortisol values are much lower (maternal/fetal ratio, 13). In this context, it would be of interest also to examine basal fetal and maternal ACTH levels and responses. Because of the small volumes of fetal blood available, it was not possible to do this in the present study.
Basal fetal ß-endorphin levels correlated with time of needling
access to the IHV (Fig. 2b), something not found in our smaller earlier
study (1). A fetal cortisol link with time became apparent
only in the current study, only in complicated procedures in which
access needling lasted more than 10 min (unpublished observations).
This supports the hypothesis that the ß-endorphin response is
controlled by the fetal pituitary and thus is more rapid than the
adrenal response; again, this should be interpreted with caution in
view of the relatively small numbers of such fetal procedures.
Ours are the first studies of human fetal HPA responsiveness in vivo. Others have looked at basal levels of cortisol and ß-endorphin, and some have examined paired maternal and fetal samples, although not in the same cohort. The finding of no relationship between basal fetal cortisol concentrations and gestational age between 17–35 weeks is similar to the results of Economides et al. (18) but in contrast to Donaldson et al. (16). However, the rise found by the latter group was predominantly in late gestation. The exact gestational period under study is important, because there could still be a rise in the last 4 weeks of pregnancy and in the first trimester (19), periods not included in the present study.
Only one previous study has compared maternal and fetal ß-endorphin plasma levels during pregnancy and has found that fetal values were higher than maternal (20). Unlike our study, Rudunovic et al. (20 did find a correlation between maternal and fetal ß-endorphin values but over a wider gestational age range, from 18–39 weeks). Our results are in agreement with those of Goland et al. (21), who similarly did not find any correlation in maternal and fetal ß-endorphin in cord blood at delivery. Radunovic et al. (22) found that basal fetal ß-endorphin increased with fetal blood sampling at the PCI but only when multiple punctures were required and time to access the vein exceeded 3 min. In contrast, we found no evidence for a ß-endorphin response to needling at the PCI in uncomplicated procedures.
In conclusion, in the human fetus, the HPA axis seems functional from midgestation. Although there is a correlation between basal fetal and maternal cortisol levels, suggesting some placental transfer, the fetal stress responses, demonstrated in response to transfusion at the IHV, are independent of those of the mother.
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Acknowledgments |
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Footnotes |
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Received January 24, 2000.
Revised June 5, 2000.
Revised September 1, 2000.
Accepted September 8, 2000.
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References |
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, PCI.